Tumor Necrosis Factor (TNF) was originally discovered as a naturally occurring secreted protein with potent cytotoxic activity on tumor cells (Carswell, E. A., et al. (1075) PNAS, 72:3666–3670; Old, L. J. (1985) Science, 230:630–632; and Beutler, B. et al. (1985) Nature, 316:552–554). TNF exerts its biological effects through interaction with high-affinity cell surface receptors which trigger specific cellular responses. Two distinct membrane TNF-receptors have been cloned and characterized. These are a 55 kDa species, designated p55 TNF-R1 and a 75 kDa species designated p75 TNF-R2 (Loetscher, H. Y. et al. (1990), Cell 61:351–360; Schall, T. J. et al. (1990), Cell 61:361; Smith, C. A. et al. (1990), Science 248:1019; Corcoran, A. E., et al., (1994) Eur. J. Biochem., 223:831–840).
Expression of TNFR1 can be demonstrated on almost every mammalian cell while TNFR2 expression is largely limited to cells of the immune system and endothelial cells. Each receptor elicits a distinct signal: the intracellular portion of TNFR1 contains a “death domain” which initiates the apoptotic pathway and NFkB activation when triggered (Tartaglia, L. A. et al. (1991) PNAS 88:9292–10296; Tartaglia, L. A. et al. (1993), Cell 74: 845–853). The role of TNFR2 is less clear as it has no direct apoptotic signaling but can activate NFkB, resulting in transcriptional activation of genes required for the inflammatory and immune response.
The two TNF receptors exhibit 28% similarity at the amino acid level. This is confined to the extracellular domain and consists of four repeating cysteine-rich motifs, each of approximately 40 amino acids. Each motif contains four to six cysteines in conserved positions. Dayhoff analysis shows the greatest intersubunit similarity among the first three repeats which contains the ligand binding section. This characteristic structure is shared with a number of other receptors and cell surface molecules, which comprise the TNF-R/nerve growth factor receptor superfamily (Corcoran, A. E., et al., (1994) Eur. J. Biochem., 223:831–840).
Crystallographic studies of TNF-alpha and the structurally related cytokine, lymphotoxin or TNF-beta (LT) have shown that both cytokines exist as homotrimers, with subunits packed edge to edge in a threefold symmetry (Hakoshima, T. and Tomita, K. (1988) J. Mol. Biol. 201:455–457; Jones, E. Y. et al. (1989) Nature 338:225–228; Eck, M. J. et al. (1992) J. Biol. Chem. 267:2119–2122).
TNF signaling is initiated by receptor clustering, either by the trivalent ligand TNF or by cross-linking monoclonal antibodies (Vandevoorde, V., et al., (1997) J. Cell Biol., 137:1627–1638). Structurally, neither TNF or LT reflect the repeating pattern of the their receptors. Each monomer is cone shaped and contains two hydrophilic loops on opposite sides of the base of the cone. The crystal structure determination of a p55 soluble TNF-R/LT complex has confirmed the hypothesis that loops from adjacent monomers join together to form a groove between monomers and that TNF-R binds in these grooves (Banner, E. W. et al. (1993) Cell, 73:431–435).
TNF plays an important role in regulating inflammation, cellular immune response, and host defense. Conversely in diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, Crohn's disease, inflammatory bowel disease and other chronic disorders of the immune system, excessive levels of TNF play a role in the pathophysiology. Indeed, blocking TNF can halt disease progression and
has led to the search for antagonists of TNF.
Several strategies at blocking TNF signaling can be employed: inhibiting TNF biosynthesis, inhibiting TNF secretion or shedding, or blocking the interaction of TNF with its receptors. A natural mechanism to down regulate TNF exists whereby the extra-cellular portion of the TNF receptor is enzymatically cleaved resulting in a freely circulating TNF binding protein or “soluble receptor” which retains its affinity for TNF but neutralizes its ability to signal through its cell surface receptor (Engelmann, H. et al. (1990) J. Biol. Chem. 265:1531–1536; Olsson, et al. (1989) Eur. J. Haematol. 42:270–275; Seckinger et al. (1990) Eur. J. Immunol. 20: 1167–1174). In cases such as autoimmune disease and chronic inflammation excessively high levels of TNF overwhelms the ability to self-regulate.
The therapeutic use of soluble TNF receptors has been proven to be an effective way to block TNF signaling. For example, ENBREL®, a soluble bivalent form of TNFR2 fused to a human immunoglobulin fragment (Fc) is used for the treatment of rheumatoid arthritis and psoriasis. Soluble TNFR1-Fc has also been shown to effectively block TNF-mediated effects in animal models but has not been approved for use in humans (Lenercept) due to immunogenicity concerns (Christen, U. et al Human Immunol. 60:774–790, 1999).
While protein engineering techniques resulting in loss-of-function (i.e. random mutagenesis) have defined regions of TNF-TNFR interaction, no successful gain-of-function has been engineered into soluble TNFR. That is, there are no known designs of a less immunogenic soluble TNFR1 protein with an enhanced ability (relative to wild-type) to block TNF-mediated effects.
Therefore, a need still exists to develop more potent, less immunogenic TNF-receptor antagonists for use as therapeutic agents. Accordingly, it is an object of the invention to provide proteins with TNF-receptor antagonist activity and nucleic acids encoding these proteins for the treatment of TNF related disorders.